An open-source programmable interface for sensory–motor biotests with zebrafish (Danio rerio). The figure demonstrates the components and functionality of the developed interface for sensory–motor biotests with zebrafish, highlighting its potential applications in behavioral research and biotest development. (A) Overview of the system architecture that enables programmable control and actuation of three independent stimuli in sensory–motor behavioral biotests. (B) Open-source control software with an intuitive GUI that allows programming of the ON/OFF duration, sequence, and number of stimulus delivery cycles for each of the three stimuli. The software also supports saving of stimulus programs for reproducible and standardized biotests. (C) The hardware interface consists of an Arduino Pro Micro microcontroller, custom PCB with three NPN transistors, and internal wiring. Only the USB cable, 12 VDC power connector, and connectors for the three stimuli channels are visible to the end user in the assembled state. (D) Schematic representation of experimental validation of the tap–startle mechanical stimulus using larval stages of zebrafish at 7 dpf. Larvae were contained in a custom fabricated 48-well plate with one larva per well. The mechanical tap stimulus was delivered in 1-minute cycles using a simple push solenoid readily available in hobby electronic stores. (E) Consistent, strong, and reproducible responses were observed during multiple 1-minute stimulation cycles (red lines). Animal tracking and data analysis were performed using EthoVision XT 16 software (Noldus Information Technology, The Netherlands) equipped with a multi-arena detection module. The figure was created using www.BioRender.com. dpf, days post-fertilization; GUI, graphical user interface; NPN, Negative–Positive–Negative; PCB, printed circuit board; VDC, volts direct current.